Volume 79, Issue 4, Pages 423-431 (February 2011) Angiotensin II diminishes the effect of SGK1 on the WNK4-mediated inhibition of ROMK1 channels  Peng Yue, Peng Sun, Dao-Hong Lin, Chunyang Pan, Wenming Xing, WenHui Wang  Kidney International  Volume 79, Issue 4, Pages 423-431 (February 2011) DOI: 10.1038/ki.2010.380 Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 1 Angiotensin II (AngII) inhibits ROMK1 channels. (a) A western blot showing the expression of type 1 Ang II receptor (AT1R) in HEK293T cells and the rat kidney. (b) Whole-cell Ba2+-sensitive K currents in HEK293T cells transfected with green fluorescent protein (GFP)-ROMK1 in the absence of AngII (control) and in the presence of 100 nM AngII and AngII+losartan (20 μM), respectively. K currents were measured with the perforated whole-cell patch clamp from -100 to 60 mV at a step of 20 mV. (c) A bar graph summarizes the inhibitory effect of AngII on ROMK1 channels at different concentrations in the absence of or presence of losartan (20 μM). K currents were measured with perforated whole-cell patch clamp at -100 mV in HEK293T cells transfected with GFP-ROMK1. I (pA), currents (pA). Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 2 Inhibition of protein kinase C abolishes the inhibitory effect of angiotensin II (AngII). A bar graph summarizes the effect of AngII on ROMK1 channels in the absence of or in the presence of calphostin C (100 nM) or GF109203x (5 μM). HEK293T cells transfected with green fluorescent protein-ROMK1 were pretreated with calphostin C or GF109203x. The K currents were measured with the perforated whole-cell recording at -100 mV. Asterisk (*) indicates a significant difference from the control value. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 3 Angiotensin II (AngII) stimulates c-Src phosphorylation and tyrosine phosphorylation of ROMK1. (a) A western blot showing that treatment of HEK293T cells with AngII for 20–30 min increases the tyrosine phosphorylation of c-Src on tyrosine residue 416. (b) A western blot demonstrating the effect of AngII on tyrosine phosphorylation of c-Src on tyrosine 416 in the HEK293T cells pretreated with calphostin C. A western blot shows the effect of AngII (100 nM) on the tyrosine phosphorylation of ROMK1 in cells transfected with green fluorescent protein (GFP-ROMK1) (c) or with GFP-R1Y337A (d). ROMK1 proteins were harvested by immunoprecipitation with anti-GFP antibodies and tyrosine phosphorylation of ROMK1 was detected with PY20. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 4 Inhibition of protein tyrosine kinase abolishes the effect of Angiotensin II (AngII). (a) The whole-cell recording demonstrates the effect of c-Src and AngII treatment on K currents in HEK293T cells transfected with green fluorescent protein-ROMK1 with or without herbimycin A. (b) A western blot showing the expression of c-Src in HEK293T cells transfected with or without exogenous c-Src. (c) A bar graph summarizes the effect of c-Src expression or 100 nM AngII treatment on ROMK1 channels in the presence of herbimycin A (1 μM). (d) Effect of AngII (100 nM) on K currents in HEK cells transfected with R1Y337A. The K currents were measured with perforated whole-cell patch recording at -100 mV. Asterisk (*) indicates a significant difference from the control value. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 5 Downregulation of endogenous c-Src abolishes the effect of angiotensin II (AngII). (a) The whole-cell recording demonstrates the effect of downregulating c-Src with small interfering RNA (siRNA), increasing K currents and abolishing the effect of AngII on ROMK1 channels in HEK293T cells transfected with green fluorescent protein-ROMK1. (b) A western blot showing the effect of c-Src siRNA on the expression of c-Src, protein kinase Cε (PKCε), AngII type I receptor (AT1R), and actin in HEK293T cells. (c) A bar graph summarizes the effect of siRNA of c-Src on AngII-induced inhibition of ROMK1 channels. The K currents were measured with perforated whole-cell patch recording at -100 mV. ‘#’ Indicates significance from rest of groups. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 6 Angiotensin II (AngII) modulates the effect of serum–glucocorticoids-induced kinase 1 (SGK1) and with-no-lysine kinase-4 (WNK4) on ROMK channels. (a) Recording showing the Ba2+-sensitive K currents measured with perforated whole-cell patch clamp at -100 mV to 60 mV under different conditions. (b) A bar graph summarizes the experiments in which the K currents were measured at -100 mV in HEK293T cells transfected with green fluorescent protein-ROMK1Y337A (R1Y337A), WNK4+R1Y337A, SGK1+WNK4+R1Y337A, and SGK1+R1Y337A in the presence or absence of 100 nM AngII. Asterisk (*) indicates the significant difference (P<0.05). Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 7 Inhibition of PTK abolishes the effect of AngII on ROMK. (a) A bar graph summarizes the effect of 50 nM angiotensin II (AngII) on ROMK channels in the cortical collecting duct of rats on a low Na diet for 1 week in the presence or in the absence of herbimycin A (Herb A). The statistical significance is indicated by ‘#’ (P<0.05) and ‘*’ (P<0.01). (b) A channel recording showing the effect of AngII on ROMK channels in the cortical collecting duct of a rat on a low Na diet. Experiments were performed in cell-attached patches and the holding potential was 0 mV. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 8 A cell scheme illustrating the role of angiotensin II in regulating ROMK channels during the volume depletion. A dotted line means a diminished effect. Abbreviations: CCD, cortical collecting duct; DCT, distal convoluted tubule; SGK1, serum–glucocorticoids-induced kinase 1; WNK4, with-no-lysine kinase-4. Kidney International 2011 79, 423-431DOI: (10.1038/ki.2010.380) Copyright © 2011 International Society of Nephrology Terms and Conditions